Project summary Retinal vascular diseases are major causes of vision loss in the United States and around the world. Age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity are all associated with the growth of new blood vessels (neovascularization) into or on the surface of the retina. They are also associated with the leakage of fluid from the retinal blood vessels into the retina (edema) and with bleeding from new vessels. To better treat these disorders, we need to understand the signaling pathways that control the growth and integrity of retinal blood vessels. Over the past decade we have characterized a signaling system that controls the growth and barrier properties of retinal blood vessels. In this pathway, Norrin, a ligand synthesized by Muller glia, activates canonical Wnt signaling via its receptor [Frizzled4 (Fz4)], co-receptor (Lrp5), and co-activator (Tspan12) on the surface of vascular endothelial cells (ECs). In humans and mice, mutations in any of the genes coding for these proteins impairs retinal vascular development. The objectives of this proposal are to develop and apply new molecular technologies and genetically engineered mouse models to: (1) identify novel protein regulators of Norrin/Fz4 signaling and determine their roles in retinal vascular development and homeostasis, (2) define, on a whole-genome scale, the transcriptional and chromatin responses of vascular ECs to Norrin/Fz4 signaling, (3) discover and characterize cell surface and secreted proteins that mediate communication between vascular ECs and their pericyte and astrocyte neighbors, and (4) develop and characterize reporter mouse lines that permit simultaneous, cell-type-specific, and cellular resolution analyses of the activities of two or more signaling pathways, and then apply these lines to the analysis of signaling (including Norrin/Fz4 signaling) in developing, adult, and diabetic vascular ECs. As the retinal vasculature is very similar between mice and humans, and the Norrin/Fz4 pathway (as well as other signaling pathways such as VEGF, Notch, and TGF-beta) are highly conserved across mammals, the proposed experiments with mice should translate directly to humans. Finally, the new technologies and mouse lines that will be developed in the proposed studies will be freely disseminated to the scientific community and should have a catalytic effect on the field.